Currently, six resistance loci have been mapped in tomato . The Ty-1 and Ty-3 genes are allelic and were introgressed from S. chilense . The Ty-2 gene originated from S. habrochaites and encodes a nucleotide-binding site-leucine-rich repeat containing protein . The Ty-4 gene is located on the long arm on chromosome 3, and was introgressed from S. chilense . The recessive Ty-5 gene encodes the messenger RNA surveillance factor Pelota, and is believed to have been introgressed from S. peruvianum . Finally, the Ty-6 gene is located on the long arm of chromosome 10, and was introgressed from S. chilense . The Ty-1 is a partially dominant resistant gene that was introgressed from the S. chilense line LA1969 . The Ty-1 locus is composed of three allelic variants, and Ty–1-like alleles are present in both resistant and susceptible tomato lines . Expression analyses of these alleles revealed that the Ty–1 gene expression was significantly higher compared to that of the ty- 1 allele, and that these differences correlate with the resistant phenotype . The Ty-1 gene encodes an RNA-dependent RNA polymerase of the γ-class, but with an atypical DFDGD amino acid motif in the catalytic domain located at the Cterminus . The γ-RdRp is found in plants and fungi, but no clear function has been established for these proteins . Investigation of the Ty-1 function has shown that the accumulation of TYLCV in inoculated tissues and the long-distance movement of the virus were impaired in Ty-1 lines,suggesting a role in suppression of cell-to-cell movement . However, vertical farm equipment it was later demonstrated that TYLCV-specific small interfering RNA levels were higher in resistant Ty-1 plants compared with those in susceptible ty-1 plants .

Furthermore, the presence of these siRNA was correlated with cytosine methylation of the viral genome . This suggested that the Ty-1 gene is involved in transcriptional gene silencing . So far, little is known about the functional properties of the Ty-1 protein, and the nature of the signal transduction pathway involved in the defense response. Here, we generated cDNA libraries from samples of inoculated stems and newly emerged leaves of the near isogenic lines LA3473-R and LA3474-S, respectively. We used an RNASeq approach to identify differentially expressed genes during the resistant and susceptible responses to TYLCV infection. Finally, we cloned and sequenced the Ty-1 gene and determine some properties of the Ty-1 protein to provided further insight into how some of the newly identified DEGs participate in the TYLCV-mediated defense response. These results will be discussed in terms of the mechanism of resistance and associated signal transduction pathways.NILs of tomato with the Ty-1 gene or ty-1 gene were used for these experiments . Seeds of these NILs were obtained from the Tomato Genetics Resource Center, UC Davis. Tomato plants of both lines were grown in a greenhouse, allowed to be self-pollinated and fruits were harvested. Seeds were extracted and treated with 2.7% sodium hypochlorite for 30 min and rinsed with MilliQ water. Treated seeds were planted into sunshine mix 1 potting mix in a controlled environment chamber .To confirm the resistant and susceptible phenotypes of plants produced from increased seeds, NILs LA3473-R and LA3474-S, respectively, seedlings at the three- to five-leaf-stage were agroinoculated with cell suspensions of a strain of Agrobacterium tumefaciens containing a binary plasmid with the multimeric infectious clone of TYLCV from California by needle puncture inoculation of the stem just beneath the shoot apex .

The positive control in these experiments were seedlings of the susceptible tomato plants cv. Glamour agroinoculated with TYLCV, whereas the negative control was seedlings agroinoculated with the empty vector . Inoculated plants were maintained in a controlled environment chamber, and symptom development was assessed visually and recorded at 7, 14 and 21 d post infection based on the severity scores described in Lapidot et al. . Absolute viral DNA accumulation was quantified at 7, 14 and 21 dpi by quantitative polymerase chain reaction tests according to the protocol described by Mason et al. . Total genomic DNA was extracted from newly emerged leaves according to the method of Dellaporta et al. . A virus-specific primer pair for qPCR detection was designed to direct the amplification of an ~150 base pair fragment from the capsid protein gene of TYLCV . The specificity of this primer pair was predicted based on BLAST search , and confirmed experimentally by conventional PCR with DNA extracts of tomato plants agroinoculated with TYLCV and non-inoculated plants. The PCR-amplified TYLCV fragment was cloned into pCR-Blunt II-TOPO to generate a standard for qPCR assays. Recombinant plasmids containing the cloned PCR-amplified TYLCV fragment were quantified with a NanoDrop1000 spectrophotometer , and plasmid copy number was adjusted to 107 copies/µl with the Avogadro’s constant . Standard curves for qPCR were prepared with tenfold serial dilutions ranging from 101 to 106 copies of plasmid DNA. These standard curves were used to estimate the viral Cn for each sample. The qPCR was conducted on a QuantStudio™ 6 Flex Real-Time PCR System with 100 ng of total genomic DNA in a 20-μl reaction mix with the SsoFast EvaGreen Supermix kit . To detect the presence of the Ty-1 or ty-1 genes in the NILs LA3473-R and LA3474-S, respectively, restriction fragment length polymorphism analyses of the amplified fragments with a marker to Ty-1/ty-1 alleles were performed.

Here, leaf samples were collected from non-inoculated leaves of LA3473-R and LA3474-S plants and total genomic DNA was extracted as previously described. PCR tests were performed with the C2_At5g61510- F/C2_At5g61510-R primer pair , which direct the amplification of an ~1.0 kb fragment. PCR-amplified fragments were purified with the QIAquick gel extraction kit and digested with the five-base-cutting enzyme HinfI and the reaction analyzed by agarose gel electrophoresis.For the RNA-sequencing experiments, a cell suspension of an A. tumefaciens strain containing the multimeric infectious clone of TYLCV-[US:CA:06] or the empty vector was used for agroinoculation of seedlings of the NILs LA3473-R and LA3474-S as previously described. A total of three plants per treatment were inoculated per experiment, and the experiment was repeated three times. Tissue samples of the resistant and susceptible lines were individually collected and pooled together in each treatment from inoculated stems and, from other inoculated plants, from newly emerged leaves for a total of eight-time points . Samples were frozen immediately in liquid nitrogen. High-throughput RNA-seq library preparation was performed according to the method described in Kumar et al. . Briefly, double-stranded complementary DNA was prepared with random hexamer priming, and the resulting cDNA was fragmented, end-repaired and A-tailed. DNA barcodes for multiplexing Illumina DNA-Seq libraries were added to the cDNA fragments during adapter ligation, and the adapter-ligated cDNA libraries were enriched with 13 cycles of PCR amplification followed by size selection of ~200-500 bp fragments. A total of 96 cDNA libraries we generated, which included the eight-time points each for the resistant and susceptible lines and for the three independent experiments. Finally, barcoded libraries for each independent experiment were pooled together and sequenced on two lanes on Illumina HiSeq 2000 platform at the UC Davis Genome Center.Raw sequence data were filtered/trimmed for low-quality reads and technical sequences with Trimmomatic . Trimmed RNA-Seq reads were aligned to the high quality, non-redundant database generated from the tomato genome by the International Tomato Annotation Group with Hisat2 . The latest ITAG database was obtained from Sol Genomics Network . Feature Count was used to count the number of reads mapped to each gene . DEGs were identified with Cuffdiff, a method that estimates the relative transcript abundance . Gene expression levels were normalized with fragments per kilobase of exon per million mapped reads values, and the false discovery rate was used to determine the differentially expressed p-value threshold. In addition, an independent DEGs analysis was performed with DEseq and EdgeR methods , commercial indoor growing systems and data were visualized with Galaxy . In the present study, genes were considered to be differentially expressed only when their absolute value of log2 fold change was >1.5 and p-value was <0.05. To identify the potential function of DEGs involved in the Ty-1 resistance response to TYLCV infection, the functional classes of DEGs were identified with gene ontology enrichment analyses with PANTHER within the Gene Ontology project .

In order to investigate the transcriptional changes in tomato stems and leaves during TYLCV infection, we performed RNA-Seq experiments with inoculated stems and systemically infected leaves in the NILs LA3473-R and LA3474-S. Overall, ~1140 million read pairs were obtained for 96 libraries, with an average of ~12 million read pairs per library . The reads from these libraries were trimmed and then aligned to the tomato genome in the ITAG database. Time course comparisons of DEGs in the NILs revealed that the largest transcriptional changes in LA3473-R occurred at early stages of infection in inoculated stem tissues. A total of 797 and 660 genes were differentially expressed at 12 and 24 hpi, respectively, and dropped to 42 DEGs at 48 hpi . In the systemically infected leaves, there were substantially fewer DEGs, with 0, 44 and 10 genes differentially expresses at 7, 10 and 14 dpi, respectively . Most of the transcriptional changes observed in the LA3474-S plants occurred from 12 to 48 hpi in inoculated stem tissues and at 7 dpi systemically infected leaves. A total of 264 , 540 and 355 genes were differentially expressed at 12, 24 and 48 hpi, respectively . In systemic infected leaves, 356 genes were differentially expressed at 7 dpi, whereas 99 and 2 genes were differentially expressed at 10 and 14 dpi, respectively . Interestingly, a similar number of DEGs were induced in both resistant and susceptible responses, with 1553 and 1616 DEG in the resistant and susceptible lines, respectively. However, the percentage of upregulated genes in the susceptible LA3474-S line was higher, whereas more genes were downregulated in the resistant LA3473-R line . In order to gain insight into the mechanism or signal transduction pathway involved in resistance to TYLCV infection, we analyzed the transcriptome of resistant vs susceptible tomato plants at the eight time points during the TYLCV infection response following agroinoculation. DEGs in the resistant vs susceptible response to TYLCV infection were identified with three different statistical algorithms, and the analyses revealed 10751 genes differentially expressed by Cuffdiff, 5051 by Deseq, and 1462 by EdgeR, respectively . Moreover, a total of 679 genes and 58 lncRNAs were identified as differentially expressed in LA3473-R during TYLCV infection by all three methods . A large number of genes were differentially expressed at 24 hpi , but the largest transcriptional changes occurred at 7 dpi in systemically infected leaves, a time when TYLCV is accumulating in emerging leaves . In contrast, a total of 50 and 17 genes were differentially expressed at 10 and 14 dpi, respectively . To further study the TYLCV-mediated defense and susceptible responses in tomato during early and late infections, DEGs in protein families involved in stress responses in plants were selected for further analyses . Of the DEG identified by all three methods, 12 genes were associated with protein families involved in tolerance to abiotic stress response and plant-pathogen interactions, such as WRKY transcription factors, nucleotide binding site-leucine rich repeats proteins, receptor-like protein kinases , leucine-rich repeat receptor kinases and chloroplast proteins. In comparisons between resistant and susceptible responses to TYLCV infection, the mannose-1- phosphate guanyltransferase was upregulated 3.1-fold at 48 hpi, the heavy metal transport/detoxification protein upregulated 5.4-fold at 10 dpi and the WRKY transcription 46 upregulated 5.8-fold at 14 dpi . Comparisons between susceptible and the control revealed that Ycf68 chromosomal protein was upregulated 4.1-fold at 14 dpi; whereas the WRKY transcription factors 45 , 46 and 55 , two NBS-LRR genes , two LRR-RKs genes and one RLK gene were downregulated 1.6 to 6.7-foldat 14 dpi . Additionally, two lncRNAs were downregulated 2.5 to 4.0- folds at 14 dpi in the susceptible response to TYLCV infection . For a better understanding of the transcriptional responses to TYLCV infection in LA3473- R plants and to reveal putative functions of DEGs, GO enrichment analyses were performed with PANTHER and the tomato reference genome sequence. In these analyses, >80% of the DEGs, respectively, were annotated. Within the biological process class, the majority of DEGs belong to the category of cellular processes, metabolic processes and biological regulation .